JP7319861B2 - Microfluidic system and respective methods for digital polymerase chain reaction of biological samples - Google Patents

Description

The present invention relates generally to the technical field of sample analysis, such as chemical analysis of chemical or biochemical reactions, and more specifically to the technical field of high-throughput analysis of biological samples. More particularly, the present invention relates to microfluidic systems for digital polymerase chain reaction (dPCR) of biological samples. More particularly, such systems are e.g. in the form of wells or microwells, respectively, which serve as reaction sites for chemical or biological reactions of at least one biological sample provided therein. , a microfluidic device with channels in fluid communication with an array of reaction areas, also called partitions, implemented as reaction chambers or reaction vessels, the system being as fast and reliable as possible in a microfluidic device. of thermocycling temperature profiles can be achieved. Furthermore, the invention relates to respective methods for dPCR of biological samples in such microfluidic systems.

In the field of diagnostic technology for chemical analysis of chemical or biochemical reactions, multiple different chemical analyzes are performed on one or more test samples on the same - preferably disposable - microfluidic device, thereby It is a goal to be able to provide a method of independently analyzing one or more test samples with multiple different reagents during a single analytical process. As such a test sample, a biological sample is usually used, e.g. is obtained. Accordingly, the terms "sample" and "biological sample" refer to materials that may potentially contain a component of interest, and samples include blood, saliva, ocular fluid, cerebrospinal fluid, sweat, urine, stool, semen, It is specifically contemplated that a sample may be derived from any biological source, such as physiological fluids including milk, ascites, mucus, synovial fluid, peritoneal fluid, amniotic fluid, tissue, cultured cells, etc., containing a given antigen or nucleic acid. can be

Most of the known chemical, biochemical and/or biological chemical assays immobilize a biological sample, such as those described above, within a reaction site, perform one or more reactions on the static sample material, Including subsequent quantitative and/or qualitative analysis processes. Here, for many biological, biochemical, diagnostic or therapeutic applications, it is essential to be able to accurately determine the amount or concentration of a particular substance or compound, i.e. a component of interest, within a biological sample. is. In order to achieve this goal as accurately as possible, various methods have been developed over the years in the field of technology, such as the widely known polymerase chain reaction (PCR) method, which is a biological It is a cost-effective method of copying or amplifying small segments of DNA or RNA within a sample that allows in vitro synthesis of nucleic acids within the sample such that DNA segments can be specifically replicated. The development of such methods for amplifying DNA or RNA segments has yielded great benefits in genetic analysis and diagnosis of many genetic diseases, or detection of viral load.

Thermocycling, also commonly referred to as thermocycling, is utilized to provide heating and cooling of reactions in a sample within a reaction chamber for amplifying such DNA or RNA segments, and laboratory equipment including thermocyclers. is commonly used to achieve an automated procedure for PCR-based diagnostic chemical analysis, during which the liquid PCR sample is repeatedly heated and cooled to different temperature levels and maintained at different temperature plateaus for a period of time. There must be. As an example, during the course of a typical PCR run, a particular target nucleic acid must be exposed to (a) a relatively high temperature, e.g. , usually at a denaturation temperature of about 94° C. to 95° C.; For primer binding to the new DNA strand, it is cooled to an annealing temperature of about 52° C. to 56° C., and then (c) the primer binds to the new DNA strand such that the original nucleic acid sequence is replicated, e.g. It is amplified by repeated cycles of a series of steps that extend/progress using a polymerase enzyme at an extension temperature of about 72° C. to produce. Repeated cycles of denaturation, annealing, extension, usually about 25-30 cycles, exponentially increase the amount of target nucleic acid present in the sample. Currently, in order to be able to accurately maintain such temperature plateaus during thermocycling, all reaction regions can be uniformly heated and cooled to obtain uniform sample yields among the reaction regions containing the sample. , a uniform temperature distribution should be maintained over the reaction zone. Commonly known thermocycling devices, such as thermal cyclers/thermocyclers, for amplifying DNA segments, which are often referred to as sample thermostatic mounts, can be used to perform regular PCR methods. , which basically consists of a mount for receiving the sample and a heat pump attached to the mount, the heat pipe for dynamic heating and cooling of the mount and thus dynamically controlling the temperature provided to the sample. It is often provided in combination with a Peltier element used for heating and a respective heat sink thermally coupled to the Peltier element for dissipating heat, for example to the surrounding environment.

In general, there is a fundamental need to continue to make diagnostic chemical analysis faster, cheaper, and easier to perform while increasing the efficiency of traditional laboratory processes and achieving high accuracy. One particular example of the mentioned method for amplifying a DNA or RNA segment that is the focus of the present invention is the digital polymerase chain reaction method, also called digital PCR or dPCR, and as described above conventional PCR methods. and can be used to directly quantify and clonally amplify nucleic acids, including DNA, cDNA, or RNA.
Here, conventional PCR performs one reaction per sample, whereas dPCR performs one reaction within a large number of partitioned samples each provided in a large number of reaction areas, resulting in more A substantial difference between dPCR and conventional PCR lies in the method of measuring the amount of nucleic acids, as reactions are performed individually in each reaction area to allow reliable collection and sensitive measurement of nucleic acids. Accordingly, considerable efforts have been made to achieve miniaturization and integration of various chemical analysis operations in order to increase the number of parallel chemical analyzes on a single carrier device. As examples of such single carrier devices, microfluidic devices such as microfluidic chips have been developed to receive microliter or nanoliter scale samples in the form of flowable sample liquids, such as aqueous sample liquids. Provide channels and microscale reaction areas. Microliter-scale reagents, which are typically pre-filled into arrays of small wells, such as microwells or nanowells, that are provided as reaction areas on a microfluidic chip are used to contact the flow of sample liquid flowing through the channels. Each type of chemical analysis placed there depends on the reagents loaded into the array of reaction areas as well as the configuration of the flow channels and detectors, and the filling of the microfluidic chip with the sample liquid depends on the sample liquid on the chip. can be carried out by pipetting to This advanced technique allows multiple chemical analyzes to be performed simultaneously on a miniaturized scale. Most of these chemical, biochemical and/or biological chemistry assays are quantitative and/or immobilisation of biological materials such as polypeptides and nucleic acids, cells or tissues in wells, and luminescence test measurements. or directed to performing one or more reactions with the immobilized material following a qualitative analytical process. For illustration purposes, FIG. 3A schematically shows a top view of an example of a known microfluidic chip 6, and FIG. 3B shows the chip 6 of FIG. 3A in a cross-sectional view along line AA of FIG. 3A. The microfluidic chip 6 consists of a lower plate 61 and an upper plate 62, i.e. a so-called two-layer microfluidic chip, the upper plate 62 comprising inlets 63 for the introduction of liquids into the chip and outflows of liquids from the chip. and the combination of the lower plate 61, the lower layer of the chip, and the upper plate 62, the upper layer of the chip, establish a channel 65 therebetween which leads to an inlet 63. Connected to the outlets 64 , an array of microwells 66 is provided in the channel 65 on its upper side, ie inside the top plate 62 . Here, when the sample liquid is filled into the channel 65 through the inlet 63 and toward the outlet 63, when the sample liquid flows along the array of microwells, each part of the array of microwells 66 is filled with the sample liquid. .

However, when it comes to the microfluidic structure of the microfluidic device, the practical miniaturization of the volume of the reaction chamber to produce the desired small dimensions results in an increase in the surface area-to-volume ratio or undesired vaporization of the sample liquid and, in particular, Several known problems increase, such as those associated with the undesirable generation of air bubbles in liquids provided in or flowing through microfluidic devices. In the focus of the present invention, air bubbles circulating in microfluidic systems can, as an aspect, not only damage the microfluidic structure of any kind of sensor used therein, but also cause unwanted mixing of samples in adjacent microwells. Air bubbles present in the liquid within the microfluidic structure can primarily damage the biological sample of interest, causing substantial experimental error and erroneous chemical analysis results, thus causing cross-contamination and thus causing substantial experimental error and erroneous chemical analysis results. , can constitute a serious problem. For example, air bubbles can cause experimental error in chromatography columns by drying out reaction components within the reaction zone.
Also, bubbles can seriously affect the optical sensing of the reaction area and the reactions that occur there, leading to failure of the chemical analysis. Especially when performing dPCR of samples in microfluidic devices, there are several reasons: (a) when filling the microfluidic device with sample liquid and separation liquid, air bubbles can be trapped in the microfluidic device; During cycling, bubbles, such as air bubbles, may grow due to evaporation of the sample liquid (see (a)), which may initially be trapped, or (c) evaporation of the sample may generate new bubbles. tend to occur frequently.

4A-4D schematically show a cross-section of a microfluidic chip 7, inserted into a respective thermocycling instrument, for illustrative reasons regarding the generation and growth of air bubbles previously described. and shown only schematically. Microfluidic chip 7 has a similar structure to microfluidic chip 6 of FIGS. Providing a channel 75 , an array of reaction areas 76 in the form of microwells is provided in the channel 75 on the upper side, ie inside the top plate 72 . Here, the array of reaction areas 76 has already been filled with sample liquid 77 and a sealing liquid 78 has been introduced into the channels 75 to seal the sample liquid 77 inside the reaction areas and flow channels from inlet 73 to outlet 74 . 75 is fulfilled. In order to be able to achieve thermocycling of the microfluidic chip 7, the chip 7 is implemented by a thermocycling apparatus in the form of a so-called plate cycler 8, i.e. a bottom heater 81 and a top heater 81 which are heatable plates, also called hotplates. It is placed inside a thermal or thermocycler with 82 . In FIG. 4A, the microfluidic chip 7 is shown with the heaters 81 , 82 unheated and a bubble 91 , such as an air bubble, fills the microfluidic device 7 with the sample liquid 77 and/or separation liquid 78 . , it is mistakenly introduced into the channel 75 immediately below the array of reaction regions 76 .

Thus, during filling, “initial” air bubbles 91 can become trapped inside the chip 7 , eg in the sample liquid in microwells or in the sealing liquid provided in the channels of the chip 7 . As seen in FIG. 4B, where at least the bottom heater 81 is turned on to provide a heating temperature above 50° C., the already introduced bubbles 91 grow due to the evaporation of the sample liquid 77 within the array of reaction regions 76. are doing. Also, new gas bubbles 92 are created due to unwanted vaporization of the sample liquid 77 within the array of reaction regions 76 . Here, new bubbles 92 may appear and grow in any reaction area during tempering, leaving the same in channel 75 . In FIG. 4C, the evolution of bubble 91, 92 growth due to undesired vaporization of sample liquid 77 is seen, channel 75 shows bubble growth along its extension, and growing bubbles 91, 92 flow through sealing liquid 78. It gradually pushes out of channel 75 and thus exits through inlet 73 and outlet 74, respectively shown here by arrows. The growing bubbles 91, 92 thereby dislodge the sealing liquid 78 from the microwells so that the sample liquid 77 in the microwells can evaporate more easily. Also, the growing bubbles 91, 92 can spread across multiple microwells, i.e., the bubbles 91, 92 displace the sealing liquid 78 from some microwells, completely 'exposing' these microwells. can", so that the contents of one microwell can migrate to an adjacent microwell. With further heating by the thermal cycler, the bubbles 91, 92 can grow further and even merge into one large bubble 93 (see FIG. 4D), resulting in a fused and still growing bubble. 93 gradually pushes sealing liquid 78 further out of channel 75 and thus out of inlet 73 and outlet 74 as indicated by the respective arrows. 4A to 4D, and in particular FIG. 4D, the undesirable bubble generation and growth causes the sealing liquid 78 to be pushed almost completely out of the array of reaction areas 76 and thereby caused by the sealing liquid 78. It can clearly be assumed that the sealing effect is nullified. Accordingly, the reaction components of the sample liquid 77 provided within the array of reaction areas 76 are not further preserved by the sealing liquid 78 and can therefore dry out. In other words, FIGS. 4A-4D show that during thermocycling, “initial” bubbles 91 and newly appearing bubbles 92 may grow due to vaporization of sample liquid 77 provided within reaction region 76 . The bubble growth continues until the measurement area provided in the form of an array of channels 75 and/or reaction areas 76 is almost empty. Therefore, removal of sealing liquid 78 by bubbles 91, 92, 93 can cause cross-contamination between adjacent reaction regions. Also, the bubbles 91, 92, 93 can increase the shear stress of the biological sample material within the reaction region 76 as cell membranes stretch under the undesirable liquid-air interface forces thus created. be. Furthermore, as already mentioned, air bubbles can be a real problem for optical detection of the reaction area and the reactions occurring therein, leading to substantial chemical analysis failures that must be avoided by all means. Therefore, removing or avoiding air bubbles is a major problem in the art.

To date, there have been various approaches to solving the problem of air bubbles in channels of microfluidic devices. For example, as described in EP2830769A1, one solution to the bubble problem is by providing specific microfluidic structures from the inlet, such as providing slits formed along the entire edge of the substrate during filling. The first is to prevent air bubbles from being introduced into the microfluidic device, and the fluid flows from the inlet manifold through the slit into the reaction chamber along substantially the entire edge of the substrate with uniform pressure and without air bubbles. However, here, a substantial drawback of such a solution is that if such microfluidic structures cannot prevent air bubbles from entering the microfluidic device, they may end up in the reaction chamber and entrap air bubbles. can be grown by vaporization of the sample at increased cycle temperatures without any means of removing . Also, vaporization of the sample at increased cycling temperatures can generate new air bubbles, causing failure of the dPCR method. Therefore, even though the known solutions provided are somewhat effective in avoiding pre-existing air bubbles from entering the microfluidic device, the existing air bubbles or newly generated air bubbles can Cannot be processed while cycling.

Another solution to the bubble problem is known, for example, from US 2005/0009101 A1, which involves performing a number of manipulations on the sample to ultimately result in the detection or quantification of the target analyte. We describe a microfluidic cassette or device that can be used, in which a light pipe is provided to allow detection of bubbles formed in the hybridization solution or wash buffer, and a peristaltic tube is provided to remove the detected bubbles. A roller is provided in functional relationship with the compliant layer in an interactive sense, the elastomeric material of the compliant layer being used as peristaltic actuation material. The solutions provided are therefore aimed at squeezing out any kind of gas bubbles from a microfluidic device, but this requires an elastomeric fluidic tip material. However, the handling and filling of such flexible tip materials is ineffective and imprecise, and the surface modification properties and optical properties of such materials are poor compared to the commonly used non-flexible tip materials. Due to the low quality, such solutions are considered unsuitable for dPCR chips.

As a further solution to the problem of air bubbles in the microfluidic device caused by evaporation of the sample liquid, pressure can be applied to the microfluidic device, for example as described in US2005/0148066A1, to produce multiple simultaneous An apparatus for performing microchemical and biochemical reactions is disclosed. Here, the device is heat cycled in a thermal cycler with grooves across the surface and a thin microhole chip is inserted into the grooves and heat cycled to prevent evaporation of the sample in the array and to prevent bubble generation or growth. A thin thermally conductive silicon pad can be used to provide pressure and thermal contact to the surface of the microhole chip. Here, with such a solution, the real drawback is the more complex construction of the overall device and the additional construction complexity required by applying pressure to the tip.

Thus, in the technical field of dPCR chemical analysis, microfluidic systems and microfluidic systems represent improved solutions for avoiding and/or removing air bubbles in channels while maintaining or improving the thermal cycling efficiency of microfluidic systems. There is a general need to provide dPCR methods for each of biological samples.

The inventors of the present invention have been able to ascertain that air bubbles can cause serious problems with successfully completing thermocycling of a sample, thus leading to substantial chemical analysis failures. . It has also been found that previously proposed solutions are not entirely sufficient or satisfactory with respect to avoiding such bubbles. In particular, the solutions already proposed in the prior art (see above) were either too expensive or insufficient to produce reproducible test results. Therefore, the introduction of bubbles into the channels of microfluidic devices and the generation and growth of new bubbles during conditioning must be avoided, necessitating new and improved solutions. Therefore, new inventive solutions exist or emerge due to the fact that when the sealing liquid is flowed through the channel, it can reliably wash air bubbles out of the exit port of the microfluidic chip through the channel. It was developed by the inventors basically based on the idea of removing air bubbles by "flushing" the microfluidic chip with a separating fluid, ie a sealing liquid. Accordingly, the present invention addresses the above-mentioned problems of simplified and more effective avoidance and/or removal of air bubbles in channels of microfluidic devices within microfluidic systems, while improving their thermocycling efficiency. .

According to a first aspect of the present invention, a microfluidic system for dPCR of biological samples is provided, wherein a sample liquid, e.g. a polar aqueous sample solution, is provided to individual reaction areas of an array of reaction areas. The microfluidic system comprises at least one microfluidic system having an inlet, an outlet, a channel connecting the inlet to the outlet, and an array of reaction regions in fluid communication with the channel. It has a fluidic device. Here, the microfluidic device can exhibit a structure consisting of at least an upper layer and a lower layer, either the upper layer or the lower layer can provide an array of reaction areas, inlets and outlets. Channels are established between the upper and lower layers and fluidly connect with an array of reaction areas, which may be implemented in the form of microwells or nanowells, thereby making the microfluidic device, for example, a microfluidic chip. For example, the width of the entire channel, also called lane width, can be in the range of 6 mm to 7 mm, such as 6.4 mm, thereby providing about 60 to 100 wells wide adjacent to each other, i.e. laterally of the channel. Space is usually provided. Additionally, the cross-sectional area of the opening of each well can have a circular, elliptical, or polygonal shape such as a hexagon. The polygonal shape of the well openings, in particular the hexagonal shape of the well openings, allows the well openings to be placed relative to each other with a shorter distance between them, i.e. to increase the distribution density of the well openings in the channel. be able to. Thus, the number of wells in the array of wells of the plate can be further maximized. Further, the width of the well openings, including the intermediate spaces between wells, can be 60 μm≦w≦110 μm, eg 62 μm (small wells)≦w≦104 μm (large wells). Further, the microfluidic system includes a flow circuit connectable to the microfluidic device for flowing liquid through channels of the microfluidic device and a microfluidic device for supplying sample liquid to the microfluidic device, e.g., by the flow circuit. and a sample liquid source connectable to the device. Materials such as cyclic olefin copolymers (COC), cyclic olefin polymers (COP), etc. may be used as materials for the microfluidic device, ie, the device layer, with the use of COP being preferred, for example, for cost considerations. Further, the flow circuit may be implemented by a tubing system, for example, a flexible tubing system consisting of one or more flexible tubes, the tubes having, for example, an inner layer of ethylene propylene diene monomer (EPDM) rubber and potentially with an outer layer of nitrile butadiene (NBR) rubber reinforced with a synthetic mesh, or commonly EPDM, NBR, fluorinated ethylene propylene polymer (FEP), polytetrafluoroethylene (PTFE), polyvinyl chloride (PVC) , polyethersulfone (PES), fluoroelastomer (FKM), silicone, and may additionally be covered with thermal insulation.

Additionally, the microfluidic system of the present invention includes a primary sealing liquid source connectable to the microfluidic device that provides the microfluidic device with an initial or primary sealing liquid for sealing the sample liquid within the array of reaction areas. The initial sealing liquid may be a non-tempered sealing liquid, ie an unheated sealing liquid provided, for example, at or near ambient temperature. Further, the microfluidic system of the present invention includes a secondary sealing liquid source connectable to the microfluidic device for providing additional sealing liquid to the microfluidic device, and a secondary sealing liquid source connectable to the flow circuit to provide additional sealing liquid through the flow path. and pumping means configured to pump the By way of example, the pumping means of the invention may be one of a peristaltic pump, a metering pump or a syringe pump, or alternatively may comprise additional fluidic components such as valves, according to the invention. The pumping means of may be any other type of pump, such as a diaphragm pump, a wobble piston pump, a microgear pump, or the like. Further, as an example of connectivity between a microfluidic device and a connectable component of a microfluidic system of the invention, the inlet and/or outlet of the microfluidic device, respectively, may e.g. a circular fluid-tight port, e.g. in the form of a luer lock adapter, for connecting a microfluidic device to a primary sealing liquid source, or for connecting a microfluidic device to a secondary sealing liquid source; It can be implemented in the form of a single connection port.

The above combination of the sealing liquid source and the pumping means of the microfluidic system of the invention, and the possibility of pumping additional sealing liquid through the channels, allows for the thermo-thermalization of air bubbles already present in the channels or of the sample inside the microfluidic device. Any kind of air bubbles that appear during cycling can be removed from the microfluidic device by flushing the channels with additional sealing liquid. Thus, according to the present invention, the adverse effects of air bubbles on test results can be at least reduced or completely avoided, resulting in significantly improved microfluidic systems.

In other words, the present invention relates to the general technical field of PCR using wellplate-based thermocycling architectures, with a primary focus on endpoint digital PCR. More specifically, well plates are provided within microfluidic chips in the form of microwell plates or nanowell plates featuring thousands of nanowells. Here, the microfluidic chip is disposable with channels in the form of closed filling channels on the opening side of the wells connected on one side to a macroscopic filling inlet port and on the other side to an overflow outlet port. can be Disposable microfluidic chips may also include multiple arrays of reaction areas with such microwell structures and thus multiple filling channels on the open side. Usually the microfluidic chip can be pre-filled with a so-called PCR master mix, which is mixed with the target mix in the form of sample liquid. After that, the channels on the opening side of the microwells are covered with a sealing liquid, such as cover oil, to prevent the sample liquid in the microwells from evaporating during thermocycling and to prevent the sample from moving from well to well. It is filled. Here, the oil can be filled into the channels using, for example, a peristaltic pump applying a pressure of about 500 mbar. Finally, the microfluidic system of the present invention can feature different sensors to perform fill checks, correct placement checks, and the like.

According to certain embodiments of the dPCR microfluidic system of the present invention, the initial sealing liquid and the additional sealing liquid consist of the same sealing liquid material, and any sealing liquid used in the microfluidic system of the present invention can be a sealing liquid of the type Furthermore, the sealing liquid should be immiscible with the sample liquid. Such immiscible liquids may be, for example, silicone fluids, such as polydimethylsiloxane (PDMS) fluids, or oily or polymeric liquids, such as mixtures of oily and polymeric liquids. Furthermore, the initial sealing liquid and the additional sealing liquid may be provided by different sealing liquid sources or by the same sealing liquid source, i.e. the initial sealing liquid source and the secondary sealing liquid source may be from different sealing liquid reservoirs or from a common It can be implemented by a sealing liquid reservoir. Specifically, if the initial sealing liquid and the additional sealing liquid consist of the same sealing liquid material or type, the primary sealing liquid source and the secondary sealing liquid source may be implemented by one common sealing liquid reservoir, which In this case, the terms "primary" and "secondary" simply refer to the function of the common sealing liquid reservoir, i.e., the common sealing liquid reservoir primarily seals the sample inside the array of reaction areas of the microfluidic device. a secondary sealing liquid source that can serve as a primary sealing liquid source to provide an initial sealing liquid to stop the microfluidic device and to provide an additional sealing liquid to flush air bubbles from the microfluidic device channels. can function as Such a configuration may be chosen due to the fact that it reduces the structural complexity of the microfluidic system compared to providing two different sealing liquid reservoirs.

According to a further particular embodiment of the dPCR microfluidic system of the present invention, the pumping means can be controlled by the control unit to pump additional sealing liquid through the channels and to wash air bubbles out of the channels as required. . Here, the control unit can be activated manually by an operator, or automatically based on feedback signals from additional system components that detect or monitor the formation of air bubbles in the flow path, for example. The control unit may also instruct the pump means to pump additional sealing liquid through the channels based on a predetermined pattern, for example based on a thermocycling process applied to the sample liquid in the reaction area. . Here, the pumping means may advantageously be controlled to pump a predetermined amount of additional sealing liquid through the channel, the pumping means constantly, intermittently or upon detection of air bubbles in the channel. Occasionally, it can be controlled to pump additional sealing liquid through the channel.

According to a further particular embodiment of the dPCR microfluidic system of the present invention, the microfluidic system may further comprise a bubble trap connected to the flow circuit for separating air from the sealing liquid, the bubble trap , is positioned downstream of the outlet of the microfluidic device. Air bubbles washed out of the channels of the microfluidic device by the flowed additional sealing liquid can thereby be removed from the sealing liquid flowing through the flow circuit. Therefore, an optional bubble trap may be placed in the flow path of the sealing liquid provided by the flow circuit to separate air bubbles from the flowed sealing liquid. Thereby, air bubbles can be immediately removed from the microfluidic system of the invention without having to collect the sealing fluid exiting the microfluidic device and extract the air bubbles therefrom.

According to another particular embodiment of the dPCR microfluidic system of the present invention, the sample liquid may be an aqueous solution containing the biological sample and reagents required for dPCR chemical analysis, wherein the sample liquid first comprises To fill each reaction area with sample liquid, it can flow through the channel into the array of reaction areas. Thereafter, ie, after providing the array of reaction areas with the sample liquid, an initial sealing liquid flows into the channels of the microfluidic device to seal the sample liquid within the reaction areas.

According to a further particular embodiment of the dPCR microfluidic system of the present invention, the microfluidic system detects the presence or generation of air bubbles in the channel during or prior to thermocycling, if the channel allows for optical monitoring. It may further comprise detection means for detecting the presence or generation of bubbles in the microfluidic device, such as an optical imaging device, eg an optical camera, capable of detecting and/or monitoring the . Here, for example, the inside of the channel can be seen from the outside, for example by an observation window, a transparent wall of the channel, or the like. Accordingly, the detection means can be used to provide a feedback signal indicative of bubble formation in the flow path, and the control unit can be triggered based on such feedback signal. Thus, with such structures, microfluidic systems can operate automatically without the need for an operator to monitor the microfluidic device during thermocycling or the like.

To enable the thermocycling temperature profile to be provided to the sample material within the reaction areas, the microfluidic system includes a thermal mount for receiving the microfluidic device for providing the thermocycling temperature profile to the array of reaction areas. can be done. For example, a thermal structure as described in connection with any one of Figures 4A to 4D can be used, ie a so-called plate cycler can house the microfluidic device of the microfluidic system of the invention. Alternatively, providing the thermocycling temperature profile to the array of reaction areas can be performed by heating and/or cooling means for heating and/or cooling the temperature of the additional sealing liquid to the desired thermocycling temperature profile temperature. Thus, the desired thermocycling temperature profile of the contents of the reaction region is obtained by heating or cooling respectively flowing through the channels of the microfluidic device rather than by the application of heat by an external heat source thermally coupled with the microfluidic device. It can be implemented by a new method of applying heat to the sample material while the sealing liquid, ie any kind of air bubbles in the channel, can be washed away. Thus, with such a solution, not only air bubble removal but also heating and/or cooling of the biological sample within the reaction area from within the microfluidic device can be achieved. Therefore, a more direct and faster application of the thermocycling temperature profile to the biological sample can also be achieved, and the commonly known external components such as top and bottom heaters can be omitted, thus reducing the known temperature of thermocycling instruments. Structure can be simplified. Here, in order to achieve such temperature control from within the microfluidic device, rather than applying heat externally, there are several options that can be combined with each other as desired, the options being: can contain.

(a) The secondary sealing liquid source contains a reservoir with additional unheated sealing liquid to heat the additional sealing liquid as needed, i.e. flow when required for the thermocycling temperature profile. A flow-type heating means is provided downstream of the reservoir for heating the sealing liquid flowing through the mold heating means, the flow-type heating means around a portion of the flow circuit coming from the sealing liquid reservoir, connected flow heaters, etc. can be implemented in the form of temperature control means provided in the
(b) the secondary sealing liquid source includes at least one reservoir with heated additional sealing liquid and at least one reservoir with unheated or cooled additional sealing liquid, the reservoir comprising , the reservoir may be detachably connected to the flow circuit by a respective valve mechanism, such as an electronically controlled delivery valve, or the reservoir may be heated and added to achieve the desired temperature of the sealing liquid supplied to the microfluidic device. may be connected to the flow circuit by a mixing valve for mixing the sealing liquid of and additional unheated or cooled sealing liquid;
(c) the additional sealing liquid comprises a conductive material, such as a graphene material, for heating the additional sealing liquid by applying power to the additional sealing liquid as needed.

When using either (a) and (b), or a combination thereof, the pump means must be configured to be able to follow the desired dPCR cycle, i.e. an additional thermostatted sample array. Each sealing liquid must be placed and removed from it again and any additional amount of additional sealing liquid must be pumped through the channel to remove air bubbles. If option (c) is used, i.e. if the sealing liquid is electrically heated, the liquid should be a conductive liquid that provides adequate resistance to be heated while an electric current is passed through it. Here, graphene solutions are suitable for such applications. However, in such cases the sealing liquid cannot be cooled electrically, instead the reservoir or the microfluidic chip itself must be cooled if sample temperature during dPCR needs to be reduced. Furthermore, when an electrically heated solution is applied, a circulating sealing solution system is used, without the need for a reservoir that requires precise control of the dPCR cycle, for example due to a comprehensive sensor structure that monitors temperature and current in real time. can be

According to further particular embodiments of the dPCR microfluidic system of the present invention, the microfluidic system may further comprise a pressure chamber surrounding at least the microfluidic device. Here, the function of such additional pressure chambers adds to the already achieved avoidance or elimination of air bubbles generated within the microfluidic system of the present invention, whereby air bubbles cannot interfere with chemical analysis results. Guarantee. For thermocycling itself, a disposable microfluidic device containing an oil-filled port can be set to a pressure of 1 to 2 bar, eg about 1.5 bar, to further suppress bubble generation during thermocycling. Here, pressure is preferably applied inside the microfluidic device by placing the entire microfluidic device in a pressure chamber.

According to a further particular embodiment of the invention, the apparatus of the invention may further comprise at least one sensor for controlling the temperature of the biological sample received in the microfluidic device, wherein such sensor can be a temperature sensor, for example combined with a flow sensor. Therefore, by providing a respective sensor to any of the components of the microfluidic system of the invention, the temperature of the biological sample during dPCR can be closely monitored, and the heating/cooling functions of the microfluidic system can can be controlled based on the measured temperature value of the sample in order to precisely and efficiently adjust the different temperature plateaus of . Thus, such sensors, e.g. temperature sensors, allow precise control of thermocycling temperatures, such as by control algorithms, temperature sensors and other sensors are used to control the respective heating/cooling rates, / Cooling power can be varied significantly by changing the pump speed of the fluid.

According to another aspect of the invention there is provided a method for dPCR of a biological sample in a microfluidic system as described above, the method being provided in particular in the form of a microfluidic chip. successively filling the array of reaction areas with the sample liquid by flowing the sample liquid through the channels and forcing the sample liquid through the channels; and sealing each reaction area after filling the reaction areas with the sample liquid. and the subsequent steps of flowing an initial sealing liquid through the channels of the microfluidic device to push the remaining sample liquid out of the microfluidic device; applying a thermocycling temperature profile to the array of reaction areas; and D. pumping additional sealing liquid into the channels of the microfluidic device to wash out the air bubbles.

Here, the steps of applying a thermocycling temperature profile to the array of reaction areas and pumping additional sealing liquid into the channels of the microfluidic device to flush air bubbles from the channels are in the course of combined steps. can be provided, i.e., where the application of temperature to the array of reaction regions is accomplished by pumping heated additional sealing liquid through the channels, the pumping of the additional sealing liquid through the channels The sample liquid in the reaction array is heated at the same time that air bubbles are flushed out of the reaction array. Further, preferably, the dPCR method of the present invention also includes the step of chemically analyzing the biological sample provided on the array of reaction areas, thereby rendering the method a dPCR-based analytical method.

According to certain further embodiments, the step of pumping additional sealing liquid through the flow path can include pumping a predetermined amount of additional sealing liquid through the flow path on demand, wherein the additional sealing liquid is pumped through the flow path. The pumping step may be, for example, constantly pumping additional sealing liquid into the channel only when the sample liquid in the reaction region needs to be thermostated, or intermittently pumping additional sealing liquid into the channel. and/or pumping additional sealing liquid through the channel upon detecting air bubbles in the channel. Here, there is the possibility of pumping additional sealing liquid into the channel if necessary, so that air bubbles already present in the channel or any kind of air bubbles appearing during thermocycling of the sample in the microfluidic device , can be removed from the microfluidic device by flushing the channel with additional sealing liquid. Thus, the method of the present invention can at least reduce or completely avoid the adverse effects of air bubbles on test results, thereby significantly reducing the risk for dPCR of biological samples in the microfluidic system of the present invention. An improved method is provided.

According to a particular embodiment of the dPCR method of the invention, the method is such that the channel permits optical monitoring, i.e. viewing from the outside into the channel, e.g. If so, the presence or generation of bubbles in the microfluidic device can be detected and/or monitored before and during thermocycling, e.g. by detection means, e.g. in the form of an optical camera. It can further comprise monitoring and detecting occurrences. Thus, the detection means can be used to provide a feedback signal indicative of bubble generation in the channel, and the control unit can be operated based on such feedback signal, resulting in a feedback-controlled dPCR method. . Alternatively or additionally, the method of the invention can further comprise separating the air bubbles from the additional sealing liquid, for example by means of a bubble trap connected to the flow circuit downstream of the outlet of the microfluidic device. As a result, bubbles that have flowed out of the channel of the microfluidic device due to the flowing additional sealing liquid can be removed from the sealing liquid flowing in the flow circuit. Therefore, the optional step of separating the air bubbles from the additional sealing liquid by a bubble trap placed in the flow path of the sealing liquid collects the sealing liquid exiting the microfluidic device and extracts the air bubbles therefrom at a later stage. It can be useful to immediately remove air bubbles from the microfluidic system of the invention without need. Alternatively or additionally, the method of the invention can also include applying pressure to the microfluidic device, for example by means of a pressure chamber surrounding at least the microfluidic device. The functionality of such an additional pressure application step can add to the already achieved avoidance or elimination capabilities of the dPCR methods of the invention, thereby further ensuring that air bubbles do not interfere with test results. Thereby, the microfluidic device can be set under a pressure of 1 to 2 bar, for example about 1.5 bar, in order to further suppress the generation of air bubbles during thermocycling, the fluid flow acting as a flexible pressure-transmitting element. Pressure can be applied inside the microfluidic device by applying pressure to the channel sidewalls or, in such cases, by applying pressure to the entire microfluidic device, which must exhibit a certain compressibility.

According to a particular embodiment of the dPCR method of the present invention, the step of applying the thermocycling temperature profile to the array of reaction areas comprises: applying heat to the microfluidic device to provide the thermocycling temperature profile to the array of reaction areas. Controlling the temperature profile of the mount, or alternatively controlling heating and/or cooling means for heating and/or cooling the sealing liquid temperature to the desired thermocycling temperature profile temperature. Here, the step of controlling the heating and/or cooling means can include any of the following options.
(a) controlling flow-type heating means provided downstream of the secondary sealing liquid source to heat additional sealing liquid as required;
(b) controlling a valve mechanism for mixing the additional sealing liquid from at least one reservoir with heated additional sealing liquid and at least one reservoir with unheated or cooled additional sealing liquid; step to do.
(c) optionally applying a voltage to the additional sealing liquid, the additional sealing liquid comprising a conductive material such as a graphene material for heating the additional sealing liquid upon application of electrical power; step.
(d) any combination of optional steps (a) to (c) above.

The inventive microfluidic systems and respective inventive methods described above can be part of an automated processing system, such as an analytical, pre-analytical or post-analytical processing system, wherein the automated processing system processes biological samples automatically. any device or device commonly used in state-of-the-art laboratories for the processing of biological samples and operable to perform one or more processing/workflow steps on one or more biological samples It includes components and covers analytical instruments, pre-analytical instruments, and post-analytical instruments. The expression "processing step" refers to a processing step that is physically performed, such as by which certain steps of dPCR implementation are performed. The term "analysis" as used herein is performed by one or more laboratory instruments or operational units operable to perform analytical tests on one or more biological samples. Includes any processing step. In the context of biomedical research, analytical processing is a technical procedure that characterizes parameters of biological samples or analytes. Such characterization of parameters includes, for example, determination of concentrations of specific proteins, nucleic acids, metabolites, ions or molecules of various sizes in biological samples, such as those derived from humans or experimental animals. The information collected can be used, for example, to assess the effects of drug administration on organisms or specific tissues. Further analysis may determine optical, electrochemical, or other parameters of the biological sample or analyte contained in the sample material.

The above method steps can be controlled by a control unit of the above microfluidic system, which can also control any kind of actuation or monitoring of the above microfluidic system and its components, the term "control unit" as used herein means It includes a physical or virtual processing device, such as a CPU, which can also control an entire laboratory instrument or an entire workstation containing one or more laboratory instruments in the way workflows and workflow steps are executed. The control unit can, for example, be loaded with different types of application software and direct the automated processing system or specific instruments or devices thereof to perform pre-analyses, post-analyses, and analytical workflow steps. The control unit can receive information from the data management unit about which steps need to be performed on a particular sample. Further, the control unit may be integral with the data management unit, may be included in a server computer, and/or may be part of one piece of equipment, distributed among multiple pieces of equipment of the automated processing system. may The control unit may, for example, be embodied as a programmable logic controller executing a computer readable program with instructions for performing operations. Here, a user interface can further be provided for receiving such instructions by a user, and the term "user interface" as used herein refers to an application interface for interaction between an operator and a machine. Any suitable piece of software and/or hardware including, but not limited to, a graphical user interface for receiving commands from an operator as input, providing feedback, and communicating information therein. Also, the system/device may expose several user interfaces to serve different types of users/operators.

As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. . Similarly, the words "comprise", "contain" and "encompass" should be interpreted inclusively rather than exclusively. That is, it means "including, but not limited to." Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The terms "plurality", "multiple" or "multitude" refer to two or more with integer multiples, ie 2 or >2, and the terms "single" or "sole" refer to one, ie =1. Furthermore, the term "at least one" should be understood as one or more, ie 1 or >1 with integer multiples. Thus, words using the singular or plural number also include the plural and singular number respectively. Further, the terms "here", "above", "before" and "below" and similar import terms when used in this application refer to this application as a whole rather than to specific portions of the application.

Descriptions of specific embodiments of the invention are not intended to be exhaustive or to be limited to the precise forms disclosed. Although specific embodiments and examples of the present disclosure are described herein for purposes of illustration, various equivalents within the scope of the disclosure provided by the appended claims will be recognized by those skilled in the art. can be modified. Certain elements of the embodiments described above and below may be combined or substituted with elements of other embodiments. Also, in the drawings, the same reference numerals denote the same elements to avoid repetition, and parts that are easily implemented by those skilled in the art may be omitted. Moreover, while advantages associated with specific embodiments of the present disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and all embodiments necessarily include It is not necessary to show the advantages that fall within the scope of the disclosure defined by the claims.

The following examples are intended to illustrate specific embodiments of the invention. Therefore, the specific implementations described below should not be construed as limitations on the scope of the invention. It will be apparent to those skilled in the art that various equivalents, changes and modifications can be made without departing from the scope of the invention as defined by the appended claims and thus such equivalents are It should be understood that embodiments are included here. Further aspects and advantages of the invention will become apparent from the following description of specific embodiments illustrated in the drawings.

1 is a schematic cross-sectional view of a microfluidic system for dPCR of biological samples according to a first embodiment of the present invention; FIG. FIG. 2 is a schematic cross-sectional view of a microfluidic system for dPCR of biological samples according to a second embodiment of the present invention; 3A and 3B are schematic structural diagrams of microfluidic systems known in the art in top and cross-sectional views. 3A and 3B are schematic structural diagrams of microfluidic systems known in the art in top and cross-sectional views. Figures 4A to 4D show different stages of dPCR along with a schematic functional diagram of bubble growth and generation within microfluidic systems known in the art. Figures 4A to 4D show different stages of dPCR along with a schematic functional diagram of bubble growth and generation within microfluidic systems known in the art. Figures 4A to 4D show different stages of dPCR along with a schematic functional diagram of bubble growth and generation within microfluidic systems known in the art. Figures 4A to 4D show different stages of dPCR along with a schematic functional diagram of bubble growth and generation within microfluidic systems known in the art.

FIG. 1 schematically shows a microfluidic system 1 for dPCR of biological samples according to a first embodiment of the invention, with the main components provided as exemplary cross-sections or pictograms. The microfluidic system 1 shown in FIG. 1 includes a microfluidic device 2 in the form of a microfluidic chip, the device 2 exhibiting a structure similar to the state-of-the-art chip 7 shown in FIG. It consists essentially of a lower plate 21 and an upper plate 22 and provides an inlet 23 , an outlet 24 and a channel 25 connecting the inlet 23 to the outlet 24 . Furthermore, an array of reaction areas 26 in the form of microwells or nanowells is provided above the channels 25, i.e. inside the top plate 22, to allow the reaction of the reaction areas 26 to be monitored from above. Furthermore, in the state shown, the microfluidic device 2 is filled with an array of reaction areas 26 , i.e. already flushed with sample liquid 27 to be filled into each single microwell of the array of reaction areas 26 . . Such filling of the array of reaction areas 26 with the sample liquid 27 can be done at a filling station (not shown) for pre-arranging the microfluidic device 2 in the microfluidic system 1 (see not) is considered part of the microfluidic system 1 used to introduce the sample liquid 27 into the array of reaction areas 26 . Also, the initial sealing liquid 28 is already filled in the microfluidic device 2 and the initial sealing liquid 28 is applied to seal the sample liquid 27 in the array of reaction areas 26 and to seal the array of reaction areas 26 . Filling the channel 25 can also be done at a filling station (not shown) prior to placing the microfluidic device 2 in the microfluidic system 1 . Alternatively, filling the channel 25 with an initial sealing liquid 28 to seal the array of reaction areas 26 may be performed by the microfluidic system 1 itself, by means of an additional sealing liquid 29, and the reaction areas 26 The portion of the additional sealing liquid 29 that actually seals the sample liquid 29 within the array of is to be understood as the “initial” sealing liquid 28 . Here the filling station is connectable to the microfluidic device 2 for providing the microfluidic device 2 with the sample liquid 27 and the filling station (not shown) is a connectable combined function of the microfluidic system 1 The parts can be considered to be the sample liquid source and the primary sealing liquid source.

The microfluidic system 1 further comprises a flow circuit 3 connected to the microfluidic device 2, the flow circuit 3 flowing an additional sealing liquid 29 through the channels 25 of the microfluidic device 2, mainly the additional sealing liquid 29. is used to flow through the channel 25 of the microfluidic device 2 . Here, the flow of additional sealing liquid 29 is indicated by the counterclockwise arrowed circle. The additional sealing liquid 29 is a secondary liquid consisting of a conditioned reservoir 41 containing heated additional sealing liquid 29 and an uncontrolled reservoir 42 containing unheated or cooled additional sealing liquid 29 . Reservoirs 41 , 42 are connected to flow circuit 3 by means of electronically controlled delivery valves 411 , 421 , respectively, substantially derived from sealing liquid source 4 . Thus, by controlling delivery valve 411 , heated additional sealing liquid 29 may be introduced into flow circuit 3 , and by controlling delivery valve 421 , unheated additional sealing liquid 29 may be introduced into flow circuit 3 . to obtain the desired temperature profile of the additional sealing liquid 29 in the flow circuit 3 . Alternatively, the tempered reservoir 41 and the non-tempered reservoir 42 share a common mixing valve, which is connected to the flow circuit 3 and functions in the sense of a mixing faucet, already Additional premixed and temperature controlled sealing liquid 29 is introduced into the flow circuit 3 and is ready to be provided to the microfluidic device 2 .

Here, in order to provide the additional sealing liquid 29 to the channel 25, a peristaltic pump 31 acting as a pump means of the microfluidic system 1 delivers the additional sealing liquid 29 to the microfluidic device 2, its inlet 23, is provided as part of the flow circuit 3 for pumping out of the outlet 24 through the flow path 25, thereby supplying heat or cold to the array of reaction areas 26 for thermocycling of the samples in the array of reaction areas 26. as well as washing away any air bubbles from the channel 25 and the outlet 24 , ie from the microfluidic device 2 , if bubbles are generated inside the channel 25 . Here, the pump 31 can constantly push additional sealing liquid 29 through the channel 25 or only add additional sealing liquid 29 if, for example, the sample liquid 27 in the array of reaction areas 26 needs to be tempered. Liquid 29 can be intermittently pumped into channel 25 . Thus, the possibility of pumping additional sealing liquid 29 into the channel 25 as required allows existing air bubbles in the channel 25 or any kind of air bubbles appearing during thermocycling of the sample liquid 27 to be eliminated by additional sealing. It can be removed from the microfluidic device 2 by flushing the channel 25 with the liquid 29 . In order to be able to finally remove air bubbles from the entire flow system, the microfluidic system 1 includes a bubble trap 32 arranged downstream of the outlet 24 of the microfluidic device 2, in this embodiment before the pump 31. and a bubble trap 32 is used to separate bubbles from the additional sealing liquid 29 that has flowed. This allows air bubbles, if any, to be immediately removed from the microfluidic system 1 . Thus, the adverse effects of air bubbles on test results can be at least reduced or completely avoided.

FIG. 2 shows a second embodiment of a microfluidic device 1' according to the invention, which basically consists of a structure similar to that described with respect to the first embodiment shown in FIG. Here, the same reference numbers denote the same elements, and respective descriptions are omitted to avoid duplication. However, in contrast to the first embodiment, the microfluidic device 1' comprises only an uncontrolled reservoir 42 containing an additional unheated sealing liquid 29, applied to the sample in the array of reaction areas 26. A flow heater 5 is provided downstream of the reservoir 42 to condition the non-tempered reservoir 42 according to the desired thermocycling temperature profile. Here again, the reservoirs 42 are each connected to the flow circuit 3 by electronically controlled delivery valves 421, which are simply controlled to provide the desired thermocycling temperature profile to the materials in the array of reaction areas 26. but most importantly to wash away air bubbles generated in the channel 25, an additional sealing liquid 29, respectively temperature-controlled, can be introduced into the flow circuit 3 and pumped to the microfluidic device 2 by a pump 31. can be

According to another embodiment, the structure of the previous embodiment shown in FIG. 2 without flow heater 5 is known in the art and described in connection with any of FIGS. 4A-4D. It can be applied to a plate cycler 8 in which the microfluidic device 2 is provided. Thereby, the provision of a desired thermocycling temperature profile to the sample liquid 27 in the array of reaction areas 26 can be established by the plate cycler 8 and the delivery of unheated additional sealing liquid 29 into the channels 25. , if present, is only used to remove air bubbles. Furthermore, according to another alternative embodiment, the structure of the embodiment shown in FIG. Including a conductive graphene material to heat the additional sealing liquid 29 by applying a voltage to the sealing liquid 29, thereby reducing the reaction area 26 without the need for an external heater or heating reservoir of any kind. Additional heating of the sealing liquid 29 is achieved so that the desired thermocycling temperature profile can be applied to the sample material in the array.

Although the invention has been described in relation to specific embodiments thereof, it is to be understood that this description is for the purpose of illustration only. Accordingly, it is intended that the invention be limited only by the scope of the appended claims.

1 Microfluidic system (first embodiment)
1' Microfluidic system (second embodiment)
2 microfluidic device 21 lower plate/lower layer of microfluidic device 22 upper plate/upper layer of microfluidic device 23 microfluidic device inlet 231 microfluidic device inlet cover 24 microfluidic device outlet 241 microfluidic device outlet cover 25 microfluidic device channel 26 reaction areas/array of wells 27 sample liquid (filled in reaction areas)
28 initial sealing liquid 29 additional sealing liquid 3 flow circuit 31 pump 32 bubble trap 4 secondary sealing liquid source 41 regulated reservoir 411 electronically controlled delivery valve 42 unregulated reservoir 421 electronically controlled delivery valve 5 flow heater 6 microfluidic chip 61 bottom plate/bottom layer of microfluidic chip 62 top plate/top layer of microfluidic chip 63 microfluidic chip inlet 64 microfluidic chip outlet 65 microfluidic chip channel 66 array of microwells 7 microfluidic chip 71 microfluidic chip bottom plate/bottom layer of microfluidic chip 72 top plate/top layer of microfluidic chip 73 microfluidic chip inlet 74 microfluidic chip outlet 75 microfluidic chip channel 76 reaction area/array of wells 77 sample liquid (filled in the reaction area)
78 Sealing liquid 8 Plate cycler 81 Plate cycler bottom heater 82 Plate cycler top heater 91 Initial bubble 92 Newly appearing bubble 93 Merged bubble

Claims (15)

A microfluidic system (1;1′) for digital polymerase chain reaction dPCR of biological samples, comprising:
having an inlet (23), an outlet (24), a channel (25) connecting said inlet (23) to said outlet (24), and an array of reaction areas (26) in fluid communication with said channel (25). at least one microfluidic device (2);
a flow circuit (3) connectable to said microfluidic device (2) for flowing liquid through said channel (25) of said microfluidic device (2);
a sample liquid source connectable to said microfluidic device (2) for providing a sample liquid (27) to said microfluidic device (2);
to said microfluidic device (2) for providing an initial sealing liquid (28) to said microfluidic device (2) to seal said sample liquid (27) within said array of reaction areas (26). a connectable primary sealing liquid source;
a secondary sealing liquid source (4) connectable to said microfluidic device (2) for providing additional sealing liquid (29) to said microfluidic device (2);
pumping means (31) connected to said flow circuit (3) and adapted to pump said additional sealing liquid (29) through said channel (25);
said pumping means (31) for pumping said additional sealing liquid (29) through said channel (25) upon detection of air bubbles in said channel (25) for flushing air bubbles from said channel (25); ), and a microfluidic system. In a microfluidic system (1; 1') according to claim 1,
Optional sealing liquids (28, 29) are immiscible liquids, preferably optional sealing liquids (28, 29) are one or mixtures of oils and polymer liquids such as silicone liquids. and more preferably, said initial sealing liquid (28) and said additional sealing liquid (29) consist of the same sealing liquid material. In the microfluidic system according to claim 1 or 2,
Said primary sealing liquid source and said secondary sealing liquid source (4) are implemented by different sealing liquid reservoirs or a common sealing liquid reservoir, preferably said initial sealing liquid (28) is a non-tempered sealing liquid , a microfluidic system. In a microfluidic system (1; 1') according to any one of claims 1 to 3,
Said pump means (31) is controlled to pump a predetermined amount of said additional sealing liquid (29) through said channel (25), more preferably said pump means (31) is adapted to pump said additional sealing liquid (29). A microfluidic system controlled to pump liquid (29) through said channels (25) continuously, intermittently or upon detection of air bubbles in said channels (25). In a microfluidic system (1; 1') according to any one of claims 1 to 4,
Said microfluidic system (1; 1') further comprises a bubble trap (32) connected to said flow circuit (3) for separating gas from sealing liquid (28, 29), said bubble trap ( 32) is a microfluidic system arranged downstream of said outlet (24) of said microfluidic device (2). In a microfluidic system (1; 1') according to any one of claims 1 to 5,
said microfluidic system (1; 1') is used to analyze said biological sample provided in the form of said sample liquid (27) on each member of said array of reaction areas (26), and /or said inlet (23) and/or said outlet (24) are for connection between said microfluidic device (2) and said sample liquid source, between said microfluidic device (2) and said primary sealing liquid source; implemented in the form of a connection port for connection and/or for connection between said microfluidic device (2) and said secondary sealing liquid source (4), and/or said microfluidic device (2) is Consists of at least a lower layer (21) and an upper layer (22), either said lower layer (21) or upper layer (22) comprising an array of said reaction areas (26), said inlets (23) and said outlets (24). wherein said channel (25) is established between said lower layer (21) and said upper layer (22) and is in fluid communication with an array of said reaction areas (26), preferably each reaction area comprising microwells or nanowells, more preferably said microfluidic device (2) is a microfluidic chip, and/or said sample liquid (27) contains said biological sample and reagents necessary for dPCR analysis an aqueous solution, preferably first said sample liquid (27) is flowed through said channel (25) to said array of reaction areas (26), said initial sealing liquid (28) being a A microfluidic system, wherein after supplying said sample liquid (27) to an array, it is allowed to flow into said channel (25) of said microfluidic device (2). In a microfluidic system (1; 1') according to any one of claims 1 to 6,
Said microfluidic system (1; 1') further comprises detection means for detecting the presence or occurrence of air bubbles in said microfluidic device (2), preferably the detection means is an optical imaging device, and A microfluidic system, preferably an optical camera. In a microfluidic system (1; 1') according to any one of claims 1 to 7,
The microfluidic system (1; 1') further comprises a thermal mount for receiving the microfluidic device (2) for providing a thermocycling temperature profile to the array of reaction areas (26). In a microfluidic system (1; 1') according to any one of claims 1 to 7,
providing the thermocycling temperature profile to the array of reaction regions (26) is performed by heating and/or cooling means for heating and/or cooling the temperature of the sealing liquid to the desired thermocycling temperature profile temperature;
Said secondary sealing liquid source (4) comprises a reservoir (42) with an additional unheated sealing liquid (29), for optionally heating said additional sealing liquid (29), Flow-type heating means (5) are provided downstream of said reservoir (42) and/or said secondary sealing liquid source (4) comprises at least one reservoir with heated additional sealing liquid (29). (41) and at least one reservoir (42) with an additional unheated or cooled sealing liquid (29), said reservoirs (41, 42) being connected to respective electronically controlled delivery valves (411, 421) or is detachably connected to the flow circuit (3) by a valve mechanism (411, 421) such as a common mixing valve, and/or said additional sealing liquid (29) A microfluidic system comprising a conductive material, such as a graphene material, for heating said additional sealing liquid by applying a voltage on demand to the. In a microfluidic system (1; 1') according to any one of claims 1 to 9,
A microfluidic system, wherein said microfluidic system (1; 1') further comprises a pressure chamber surrounding at least said microfluidic device (2). A method of digital polymerase chain reaction dPCR of biological samples in a microfluidic system (1; 1') according to any one of claims 1 to 10, comprising:
by flowing a sample liquid (27) through said channels (25) of said microfluidic device (2), particularly provided in the form of a microfluidic chip, and forcing said sample liquid (27) through said channels (25) , successively filling the array of reaction areas (26) with the sample liquid (27);
for sealing each reaction area of the array of reaction areas (26) after the array of reaction areas (26) has been filled with a sample liquid (27) and remaining from the microfluidic device (2). flowing an initial sealing liquid (28) through the channel (25) of the microfluidic device (2) to push out a sample liquid (27);
applying a thermocycling temperature profile to the array of reaction areas (26);
and C. pumping additional sealing liquid (29) through said channels (25) of said microfluidic device (2) to flush air bubbles from said channels (25). 12. The method of claim 11, wherein
Said step of delivering additional sealing liquid (29) through said channel (25) optionally comprises delivering a predetermined amount of additional sealing liquid (29) through said channel (25), preferably , said step of pumping additional sealing liquid (29) through said channel (25) comprises continuously pumping additional sealing liquid (29) through said channel (25); intermittently pumping through the channel (25) and/or pumping additional sealing liquid (29) through the channel (25) upon detection of air bubbles in the channel (25); Method. 13. The method of claim 11 or 12, further comprising
monitoring and detecting the presence or generation of bubbles in said microfluidic device (2), preferably by means of detection means such as an optical imaging device, e.g. an optical camera; separating air bubbles from additional sealing liquid (29) by means of a bubble trap (32) connected to said flow circuit (3) downstream of said outlet (24); and/or preferably at least said microfluidic device applying pressure to the microfluidic device (2) by a pressure chamber surrounding (2) and/or analyzing the biological sample provided to the array of reaction areas (26); Method. 14. A method as claimed in any one of claims 11 to 13,
Applying a thermocycling temperature profile to said array of reaction areas (26) comprises:
controlling the temperature profile of a thermal mount receiving said microfluidic device (2) to provide a thermocycling temperature profile to said array of reaction areas (26); or adjusting the sealing liquid temperature to a desired thermocycling temperature profile temperature. controlling heating and/or cooling means for heating and/or cooling to 15. The method of claim 14, wherein
The step of controlling the heating and/or cooling means (5) comprises
controlling flow-type heating means (5) provided downstream of said secondary sealing liquid source (4) to heat said additional sealing liquid (29) as required; additional sealing liquid (29) from at least one reservoir (41) containing additional sealing liquid (29) and at least one reservoir (42) containing unheated or cooled additional sealing liquid (29). and/or optionally applying a voltage to said additional sealing liquid (29), said additional sealing liquid (29) includes a conductive material, such as a graphene material, for heating said additional sealing liquid (29) by application of a voltage.

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